Using poliovirus (PV) and its RNA-dependent RNA polymerase (RdRp), 3Dpol, as our primary model system, we, our collaborators and other picorna virologists have made a very compelling case for RdRp fidelity and the corresponding genetic diversity of the viral population as contributors to viral pathogenesis and virulence. This relationship between viral genetic diversity and viral fitness established using the PV model has now been demonstrated for a variety of virus families. In addition, we have demonstrated that the rate of nucleotide addition can be tuned and contributes to viral genetic diversity and viral fitness. Therefore, the overarching hypothesis driving our studies is that mechanisms governing RdRp speed and fidelity can be targeted genetically and chemically for development of attenuated viruses and antiviral agents, respectively. If one is to harness the full therapeutic an prophylactic potential of modulated RdRp function, however, knowledge of the physical mechanism(s) controlling RdRp speed and fidelity is needed. One goal of this application is to test and to exploit a new model for nucleotide selection that we anticipate can be applied to any RdRp. Recombination is a major contributor to viral evolution, leading to the emergence of vaccine-resistant strains and expansion of virus host range. Unfortunately, we lack sufficient knowledge of the molecular mechanism of RNA virus recombination to prevent this process from thwarting vaccine development and long- term efficacy. Therefore, an understanding of recombination is of potentially broad, practical value. For example, detailed knowledge of the mechanism of recombination may establish principles that can be exploited for development of strategies to suppress recombination and/or design of recombination-deficient vaccine strains. A second goal of this application is to create fundamental knowledge on the mechanism of RNA virus recombination using the PV model. During the next funding period, our central objectives will be: (1) Identify active-site determinants of RdRp incorporation fidelity that can be targeted for viral attenuation; (2) Exploit the promiscuity of the RdRp nascent base pair-binding pocket for antiviral therapy; (3) Elucidate a mechanism for template switching/strand transfer by PV RdRp and establish its relevance to RNA recombination by PV in cells. The proposed studies will create important knowledge that should contribute to development of strategies to treat and to prevent RNA viral infections, including those of current concern: Ebola virus, enterovirus D68, and Middle Eastern respiratory syndrome coronavirus.